Project Summary/Abstract This proposal aims to address the clinical and scientific problem of targeted drug resistance in basal cell carcinoma (BCC). BCC is the most common human cancer in the U.S., and its tumorigenesis is dependent on increased Hedgehog (Hh) signaling, where activation of Smoothened (SMO) leads to increased Gli transcription factor activity. Targeted inhibitors of SMO have been used to treat advanced or inoperable BCCs. However, over 50% of advanced BCCs develop resistance to these SMO inhibitors, rendering the targeted therapy useless. This suggests the existence of other non-canonical pathways of Gli activation. The Oro lab has recently identified one such novel, non-canonical pathway of Gli1 activation, where RhoA signaling activates myocardin related transcription factor (MRTF) and serum response factor (SRF) to bind Gli1 and induce transcription. However, a large gap in the understanding of this pathway is the mechanism of increased RhoA signaling in resistant BCCs. An initial inhibitor screen against selected overexpressed targets in resistant BCC cells has identified transforming growth factor beta (TGFB) and bone morphogenic protein 1 (BMP-1) as necessary for maintenance of Gli1 signaling. Therefore this current proposal aims to investigate the hypothesis that TGFB (Aim 1) and BMP-1 (Aim 2) function to promote SMO inhibitor resistance in BCC by activating the non-canonical Rho-MRTF- SRF-Gli signaling pathway. To assess the necessity and sufficiency of these proteins, inhibition and overexpression experiments will be conducted in vitro in BCC cell lines. Changes will be measured at the transcriptional, protein, and cell functional levels. Because bioavailable inhibitors against TGFB and BMP-1 are already available, they represent very promising candidates for new therapeutics. A preclinical BCC mouse model will be treated with these inhibitors to test their efficacy in vivo. The results of this proposal can be used to develop more effective combination therapies in the treatment of drug-resistant BCC.